The present invention relates generally to the field of sensors for the detection and investigation of proteins, pathogens and other biologically important molecules, and more particularly to a bionanotechnology method for rapidly determining the surface topology of protein molecules.
Determining the surface topology of proteins is vitally important to broad fields of science including pharmaceutical research and genomics, as well as fundamental research into biological processes. Surface topology determines how proteins interact with each other, since proteins of complementary shape tend to bind together while proteins of non-complementary shapes do not. This lock-and-key binding of proteins provides the mechanisms by which enzymes, antibodies and nucleic molecules accomplish their vital biological functions, as well as the mechanisms that enable viruses and bacteria to infect cells. Knowing the shape and surface characteristics of a protein can help explain its function in a cell. Also, knowing the surface topology of a protein permits the development of drugs and sensors that will bind with that particular protein, enabling more effective medications and diagnostic tests for the treatment of human diseases.
Understanding the surface topology of biologically significant proteins is an important step towards the development of new medicines and medical treatments. Once the shape and chemical structure of a protein is known, pharmaceutical researchers can design a complementary molecule that will fit into the surface folds of the protein and bind to it just as an antibody binds to an antigen. Then pharmaceutically active molecules can be attached to the binding molecule to deliver a specific medication to a particular cell, bacterium or virus.
Understanding the surface topology of proteins is also essential to the development of sensitive diagnostic and protein assay methods. With the rapid advances of biotechnology and genetic research, which are elucidating the key roles and the presence of particular proteins that can be characteristic of certain disease states, great emphasis has been placed on developing very sensitive assays and sensor methods capable of detecting minute quantities of biologically significant molecules. The detection and quantification of specific proteins associated with a given disease, such as a given cancer, may enable much earlier detection and treatment. Furthermore, the ability to detect nucleic acid sequences encoding particular proteins enables many beneficial medical and commercial applications, including agricultural product screening and development. However, in many applications, methods used to detect particular proteins still require an understanding of the binding sites and surface topology of the protein.
The primary method used to develop an understanding of protein surface topologies is protein X-ray crystallography. X-ray crystallography makes use of the diffraction of X-rays by protein crystals to determine the precise three-dimensional arrangement of atoms within the protein molecule. Sophisticated analysis software and algorithms permit researchers to translate the diffraction patterns into three-dimensional structural information, and from that to identify the surface topology of the protein.
Before a protein can be studied with X-ray crystallography, the protein must be isolated, purified and crystallized. Because of the molecular complexity of proteins, obtaining suitable crystals for crystallography can be quite difficult, extremely time consuming and labor intensive. In response, a number of methods and equipment designs have been developed for crystallizing proteins (see, for example, U.S. Pat. No. 5,961,934, issued Oct. 5, 1999; U.S. Pat. No. 5,643,540, issued Jul. 1, 1997; U.S. Pat. No. 5,597,457, issued Jan. 28, 1997; U.S. Pat. No. 5,419,278, issued May 30, 1995; and U.S. Pat. No. 5,096,676, issued Mar. 17, 1992, having methods for forming protein crystals suitable for crystallography) and for streamlining the effort required to identify and reproduce appropriate conditions for crystallization of proteins (see, for example, U.S. Pat. No. 5,641,681, issued Jun. 24, 1997, showing a method for obtaining conditions for growth of high quality protein crystals). A number of physical and chemical factors can impact upon protein formation and crystal growth, so that, for example, it has been proposed that protein crystals be produced in a satellite in earth orbit having zero gravity.
Thus, there is a need for a less time consuming and expensive method for determining the surface topology of proteins, to benefit medical and biological research and facilitate the development of new medications.
Furthermore, there is a need for a means to quickly identify potential binding sites on specific proteins. In many pharmaceutical and diagnostic commercial applications, only potential binding sites on the surface of a protein, rather than a detailed model of all atomic coordinates, are of interest. Identifying binding sites by current methods requires sophisticated analyses of the three-dimensional structure of the protein involving complex numerical modeling. Thus, a less expensive and more direct method for determining potential protein-binding sites would be of economic value.
There is also a need for reliable diagnostic devices to diagnose individuals who are infected, for example, by a new or rapidly mutating pathogen, such as human immunodeficiency virus (HIV), influenza virus, or malaria, or other disease states that have evaded simple diagnostic tests, such as certain forms of cancer or pre-cancerous conditions.
In one embodiment, the invention provides a method for discovering protein adsorption sites on a surface, comprising: providing a test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters (one Angstrom) to about 10xe2x88x928 meters (10 nanometers) in width, height, depth and spacing; exposing the test surface to a solution of a substantially purified protein, the solution remaining in contact with the surface sufficiently long to enable protein molecules to adsorb to adsorption sites; removing the solution with unadsorbed protein molecules from the test surface; and identifying the protein adsorption sites by detecting the presence of adsorbed protein molecules, to locate protein molecules adsorbed to the test surface.
A related method further includes removing the protein molecules adsorbed to the test surface, and measuring the surface topology of the identified adsorption site.
In accordance with a related embodiment, the method further comprises analyzing statistically the surface topology of a statistically significant number of identified adsorption sites to determine a most probable adsorption site topology. Measuring the surface topology further comprises using one or more of the group consisting of: a microcantilever, an atomic force microscope, a scanning tunneling microscope, a magnetic resonance force microscope, a thermomechanical atomic force microscope, a multi-tip atomic force microscope, and microparticles coupled to protein molecules adsorbed to the test surface. The method in various embodiments further comprises storing the most probable adsorption site topology in a computer database.
Another related method includes detecting the presence of adsorbed protein molecules using a microcantilever. Yet another related method includes detecting the presence of adsorbed protein molecules using, for example: an atomic force microscope; a scanning tunneling microscope; a magnetic resonance force microscope; a thermomechanical atomic force microscope; is a multi-tip atomic force microscope; or coupling a microparticle to protein molecules adsorbed to the test surface.
Another related method includes using a plurality of different surface coatings deposited on the test surface, each coating having a characteristic resiliency, and further comprising determining which of said surface coatings is deposited on the identified adsorption sites by measuring the resiliency of the deposited surface coatings using an atomic force microscope.
Another related method includes using a plurality of different surface coatings deposited on the test surface, each coating having a characteristic conductivity, and further comprising determining which of the surface coatings is deposited on the identified adsorption sites by measuring the conductivity of the deposited surface coatings using a conducting atomic force microscope.
Another related method includes providing a test surface wherein the features are not randomly distributed, and the features have a width, a height, a depth and a spacing in an identical pattern distributed regularly on the test surface.
In another embodiment, the present invention provides a method for determining a topology of protein binding sites, comprising: providing a test surface having a surface topology having a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing; depositing a solution containing substantially a single type of protein molecules on the test surface, and permitting the solution to remain sufficiently long to enable the protein molecules to adsorb to protein binding sites on the test surface; removing unadsorbed proteins from the test surface; using an atomic force microscope for identifying a location of the adsorbed protein molecules on the test surface; and determining the topology that is complementary to the surface topology measurements of the identified adsorption sites. This method can further comprise, prior to depositing a solution of proteins on the test surface, using an atomic force microscope to obtain a plurality of surface topology measurements of the test surface, and recording the surface topology measurements in a computer database. This method can also further comprise analyzing a statistically significant number of the surface topology measurements of the identified adsorption sites, to determine a most probable adsorption site topology and a most probable complementary protein surface topology of the single type of protein.
In another embodiment, the present invention provides a method for determining attachment areas on a surface of a species of a micro-organism, comprising: providing a test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing; depositing a sample containing a plurality of units of the micro-organism on the test surface and permitting the sample to remain in contact with the test surface sufficiently long to enable surface proteins on the micro-organism to adsorb to areas on the test surface; removing unadsorbed units of the micro-organism from the test surface; and identifying the location of attachment areas on the test surface by using a microscope by locating micro-organisms adsorbed to the test surface. The method includes an embodiment wherein the units of the micro-organism are selected from the group of virions, bacterial cells and spores.
In another embodiment, the invention provides a method for determining a topology of potential protein binding sites for a surface of an envelope of a pathogen, comprising: disrupting the pathogen, removing soluble components, and solubilizing envelope proteins of the pathogen, to obtain a sample of surface proteins of the pathogen; providing a test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing; depositing the sample of surface proteins on the test surface and permitting the sample to remain in contact with the surface sufficiently long to enable proteins to adsorb to features on the test surface; removing unadsorbed proteins from the test surface; and identifying sites on the test surface by detecting the locations of adsorbed pathogen surface proteins, to locate sites on the test surface for adsorption of proteins the pathogen envelope.
In accordance with related embodiments, the method includes: identifying the adsorption sites with an atomic force microscope, to obtain surface topology measurements of the identified adsorption sites; analyzing statistically the surface topology measurements of a statistically significant number of the identified adsorption sites, to determine a most probable adsorption site topology; storing the most probable adsorption site topology in a computer database; determining a complementary topology to the most probable adsorption site topology; or storing the complementary topology in a computer database. In accordance with a related embodiment, the pathogen is a virus or a bacterium. Further, the absorbed surface proteins can be further characterized by eluting from the test surface, for example, wherein eluting protein from the test surface is characterizing the protein molecule by laser desorbing and analyzing a time-of-flight characteristic, for example, wherein analyzing the time-of-flight characteristic comprises obtaining a molecular weight. Obtaining the molecular weight further comprises comparing the molecular weight to protein sequence data predicted from a sequence of a nucleic acid of the pathogen.
In yet another embodiment, the invention provides a method for rapidly developing a diagnostic device for a disease, comprising: obtaining a first set of biological samples from each of a statistically significant number of individuals having a disease, and a second set of biological samples from each of a statistically significant number of disease-free individuals; providing a test surface having a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing; depositing each of first set of the biological samples on the test surface, and permitting each sample to remain sufficiently long to enable proteins from each of the first set to adsorb to adsorption sites; removing unadsorbed proteins from the test surface; identifying adsorption sites on the surface from each of the samples of the first set by detecting the presence of an adsorbed protein, to locate proteins adsorbed to the test surface; repeating the method for each of the samples from each of the second set; and identifying a pattern of protein adsorption sites that is different in samples of the first set having the disease compared to samples of the second set lacking the disease.
Accordingly, the disease can be a type of cancer or a precancerous condition; further, the disease can be infection by a human immunodeficiency virus. In related embodiments, the invention provides a diagnostic device having a protein adsorption pattern discovered by any of these methods, for example, a diagnostic device replicating a surface topology of a protein adsorption site, using test surface topology measurements obtained by such a method. Further, related embodiments include a composition which is a protein associated with a disease, the protein being identified using any of the related embodiments of the methods.
In yet another embodiment, the invention provides a method for determining potential molecular attachment areas on a surface of a pathogen comprising: providing a test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing; depositing a solution containing a preparation of units of the pathogen on said test surface and permitting said solution to remain sufficiently long to enable surface proteins on the pathogen to adsorb to adsorption sites on the test surface; removing unadsorbed pathogen from the test surface; and identifying adsorption sites on said test surface by using a microscope to locate units of the pathogen adsorbed to said test surface. For example, the units of the pathogen are virions or bacteria.
In yet another embodiment, the invention provides a diagnostic device, comprising: a test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing; a pattern of protein adsorption sites of known specificity on the test surface; and a reader capable of detecting proteins adsorbed to the pattern protein adsorption sites of known specificity. A related embodiment is provided, further comprising a fluidics cell, the fluidics cell having a fluid inlet, a fluid outlet, and a wall structure coupled to the test surface. For example, the fluid inlet and the fluid outlet are micropipettes coupled to the wall structure.
In yet another embodiment, the invention provides a protein assay apparatus, comprising: an atomic force microscope, the atomic force microscope having a multi-tip microcantilever array sensor; a positioning table coupled to the atomic force microscope and configured to position a sample for inspection by the multi-tip microcantilever array; and a test surface coupled to the positioning table, the test surface having a surface topology comprised of a random distribution of randomly shaped features of a size from about 10xe2x88x9210 meters to about 10xe2x88x928 meters in width, height, depth and spacing. In accordance with related embodiments, the protein assay apparatus further comprise a fluidics cell, the fluidics cell having a fluid inlet, a fluid outlet, and a wall structure coupled to the test surface. The fluid inlet and the fluid outlet can be micropipettes coupled to the wall structure; the positioning table can be a turntable, for example, the turntable is a miniature computer drive platform. The protein assay apparatus can further comprise a fluidics cell coupled to the test surface, the fluidics cell having a fluid inlet and a fluid outlet and comprising a wall structure coupled to the test surface.